Dual roles of BK Polyomavirus in promoting urothelial carcinoma progression via regulating CLDN1

Uncontrolled productive infection of BK polyomaviruses (BKV) in immunocompromised patients was reported to result in serious diseases, especially renourinary malignancies. However, the mechanism of BKV as a role of human carcinogen is still unknown. In this study, we showed that there is a significant association between BKV infection and metastasis of urothelial carcinoma (UCA). BKV-infected tumor tissues exhibit invasive histologic phenomena with vascular invasion and myometrial invasion. Then we identified that BKV promotes UCA invasion in a mode of dual regulation of tumor cells (TCs) invasion and endothelial cells (ECs) adhesion by encoding miRNAs. In cancer cells, BKV-B1-miR-5p promotes cell motility and invasiveness by directly targeting CLDN1. Moreover, exosomal-BKV-B1-miR-3p derived from BK-infected BC cells would be transferred to ECs and increase its adhesion to tumor cells by switching on the CLDN1 enhancer, which subsequently destroyed endothelial monolayers and increased permeability. In a human urothelial cancer metastasis mouse model, BK-inoculated cells exhibited higher incidence of vascular leakage and liver colonization. However, the vascular leakage and liver metastasis could be reduced when knocking down miRNAs in BK-inoculated cells. Our research delineates the bifunctional impact of BKV-encoded microRNAs on the expression of CLDN1 within both TCs and ECs, which orchestrates the establishment of a pre-metastatic niche in UCA. Supplementary Information The online version contains supplementary material available at 10.1186/s40364-024-00564-2.

six cycles of freezing the infected cells and supernatant at − 80 °C and thawing at 37 °C to remove cell debris.Then the viral lysates were centrifuged at 10,000× g for 15 min at 4°C, and supernatant were aliquoted and store at -80°C.All BKV stocks were tittered by endpoint dilution in Vero cells.

BKPyV infections and drug treatments.
For cell counting assay, 6-well plates seeded 1 × 10 6 cells per well were infected with BKV at an MOI of 0, 1, 2, 4. Excess viruses were removed after 2 h of incubation.
After 48 hours incubation, cells were digested by 0.25% trypsin (Gibco, USA) and centrifuged at 200g 5min.After using 1ml PBS to resuspend cells in each group, we performed cell counting using automated cell counter.The assay was repeated three times.Subsequently, HTB-9 cells seeded into 6-well plates (1 × 10 6 cells per well) were infected with BKV at an MOI of 1, unless indicated otherwise.Excess viruses were removed after 2 h of incubation.The cells were washed once with PBS (Corning).We included a DMSO control at a corresponding concentration in the drug treated experiments.

Scratch wound healing assay and transwell assays
A 12-well plate was seeded to confluence with 2×10 5 cells for wound healing assay.
Scratch wounds were made using a 200 µl pipette tip.Then, medium without fetal bovine serum was used to culture the cells.Images were captured at 0h, 24h, and migration rate was calculated by ImageJ.The Transwell Permeable Supports with 8 μm pore (Corning, USA) were used to perform the cell invasion assays.The basement membrane was pretreated with 20 μg matrigel matrix (Corning, USA) and 1.5×10 5 cells/cm 2 were seeded into upper chambers.After 24 h, the transwell was fixed with 4% PFA for 15 min and stained with hematoxylin for 15 min and the cells on the upper side of basement membrane were gently removed by cotton swabs.Four random microscopic fields (magnification, 20×) were imaged for the cells on the lower side of basement membrane and the cell number was counted using ImageJ.

Dual luciferase reporter assays
After transfection of plasmids for 48 hours, the cell extracts were prepared and assayed using a Dual Luciferase Reporter Assay Kit (MCE, China) according to the manufacturer's procedures.For transfection efficiency, Renilla luciferase was used to normalize, and the ratio of firefly/Renilla luciferase activities defined the relative activity of the enhancer region and the binding of 3'UTR or predicted enhancer region.
The assay was repeated three times.

Endothelial transendothelial invasion assays
The Transwell Permeable Supports with 8μm pore were seeded separately in the top well with 1 × 10 6 /cm 2 HUVEC cells and in the bottom well with 1.5 × 10 5 /cm 2 HTB-9 cells (HTB-9 BK/HTB-9 CON), and cocultured for 72 h.Then moved the upper chamber to another well with basal medium.The top well was seeded with 1.5 × 10 5 /cm 2 GFP-labeled HTB-9 cells, after 24 h, the cells on the lower side of the chamber were imaged and counted.
By ultracentrifugation, exosomes in cell culture medium were purified 18 .Transmission electron microscopy (TEM) and Western blotting for CD9, CD63, and TSG101 were used to identify the collected exosomes.For EM, 4% paraformaldehyde with equal volume was used to fix PBS containing exosomes, applied to the formvar-coated grids and adhered for 20 min, PBS was blotted up by filter paper gently, the grid was infiltrated by 1% uranylformate solution for 1 min and repeated two times.Images were acquired by a Transmission Electron Microscope.To estimate the particle size of exosomes, NTA was performed by NanoSight NS300 (Malvern, UK).

Exosome uptake by HUVECs
Exosomes were labelled with a green fluorescent dye (PKH67; Sigma-Aldrich, St. Louis, MO, USA) as previously described 19 and later incubated with HUVECs at 37°C for 24 hours.These cells were subsequently washed with PBS and fixed in 4% paraformaldehyde for 15 minutes.Fixed cells were washed with PBS, and nuclei were stained with DAPI (0.5 μg/mL; Invitrogen) and cytoskeleton were identified by immunofluorescence staining of β-actin.Confocal microscopy was applied to detect the signals in cells.

Animals and in vivo assays
For generating Transplanted tumor models of urothelial carcinoma in nude mice, approximately 5 × 10 6 HTB-9 cells were injected Subcutaneously in the flank of each 6-week-old male Balb/c nude mouse.After 3 weeks' feeding, the volume of initiated-tumors were measured.By using (X2Y)/2, where X = tumor width and Y = tumor length, the tumor size was calculated.To gain the tumors for further analyses, mice were sacrificed after 7 weeks.For histopathologic analyses, the standard procedure was performed.For metastasis assays, 2 × 10 6 cells resuspended in 200 μl sterile PBS were injected into the lateral tail veins of each 8-week-old male Balb/c nude mouse.After 10 weeks, mice sacrificed the livers were collected to count the metastatic nodules on surface, and then these tissues were fixed in 4% paraformaldehyde for further hematoxylin and eosin staining.

CUT&TAG sequencing and CHIP-qPCR assay
We used 2×10 5 HUVECs incubated with extracted exosomes for 48 hours to perform this assay.Cells were immobilized on Concanavalin A-coated beads and incubated with H3K27ac antibody or IgG control in primary antibody buffer for 2 hours at room And for CHIP-qPCR assay, after 48 hours incubation with extracted exosomes, the cells were washed twice using PBS and fixed in 1% formaldehyde for 15 min at room temperature.Then the formaldehyde was quenched by 0.125M glycine solution, and the cells were harvested and resuspended in lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.5% NP-40,10 mM KCl) containing protease inhibitor cocktail (CST).Nucleus extracts were gained and dissolved in nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 0.3% SDS, 10 mM EDTA and 1× cocktail).After sonication, the cell extract was incubated with antibody against H3K27ac at 4•C for 16 h and Protein A Dynabeads (CST).Further immunoprecipitated DNA purification and qPCR assay were conducted according to the instruction of CHIP-kit obtained from CST.

CRISPR/Cas9 system
To delete the predicted enhancer region containing bkv-miR-B1-3p locus, we purchased 6 designed gRNA for performing CRISPR/Cas9 on the certain region from HanBio, China.Through flow cytometry, transfected cells were separated to 96-well plate, cultured in 5% CO2 at 37•C for 2 weeks, and then transferred into 24-well plate to obtain monoclonal cells.To screen the cells with efficient deletions, PCR products of the monoclonal cells were sent to Sanger sequence.

Statistical Analysis
Data analysis were conducted using GraphPad Software (GraphPad Holdings, USA).
Comparisons between two groups were analyzed by unpaired Student's t-test.
One-way ANOVA with Tukey post hoc tests was used for comparisons between multiple groups; and two-way ANOVA was used for comparisons between multiple groups when there were 2 experimental factors.Fisher's exact test was used to investigate the correlations between BKV LTAg staining results and distant metastasis condition or tumor pathological grade.A p-value<0.05 was considered statistically significant.

For
endothelial permeability assay, 6-week-old male Balb/c nude mouse were injected with 5 × 10 5 HTB-9 cells in 250 μl sterile PBS vial tail vein.6 hours later, 200 μl 2.5 mg/ml FITC-labeled 40-kD dextran (Sigma-Aldrich, USA) 10 min before sacrificing.For antagomir delivery, 3nmol antagomir-5p and 3nmol antagomir-3p in 200μl sterile PBS were injected into the lateral tail veins of each 4-week-old male Balb/c nude mouse 3 times per week for 4 weeks.All animal experiments were approved by the Animal Welfare & Ethics Committee of Shanghai Public Health Clinical Center Laboratory and in compliance with ethical guidelines and procedures.Immunohistochemistry and Immunofluorescence staining analysisOn paraffin sections, immunohistochemical staining was performed.Antigen retrieval was conducted by EDTA (pH 9.0) in steam bath, then Sections were washed with TBS three times after cooling, and incubated in 3% H2O2 for 30 min.After that, antigen was blocked by 10% goat serum for 30 min.With primary antibodies at 4 °C overnight, the sections were labeled.Goat-anti-rabbit antibody was added to the sections on the next day.DAB was used to reveal antibody binding states.Prepared slides were photographed by ECLIPSE E600 microscope with an attached Digital Sight Camera (Nikon, Tokyo,Japan).For Immunofluorescence staining, as primary antibodies, rabbit anti-CD31 antibody (CST, 1:200), rabbit anti-SV40(CST, 1:200) were used and were separately detected by Alexa Fluor 488 donkey anti-rabbit secondary antibody (1:1000; Invitrogen) and Alexa Fluor 594 donkey anti-mouse antibody (1:200, Invitrogen).For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI) was used.RNA extraction, qRT-PCR By qRT-PCR, BKPyV DNA loads were determined.The expression level of both BKV encoded large T antigen gene and VP1 gene demonstrated BKV DNA loads.Total RNA fractions were extracted with TRIzol reagent (Sigma-Aldrich).By a NanoDrop apparatus, the concentration and purity of RNA were tested.RNA with amount of 1μg was reversely transcribed into cDNA by PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan).Using Hieff qPCR SYBR Green Master Mix (high Rox) (Yeasen, China) according to the manufacturer's instruction on An ABI QuantStudio5 platform, quantitative real-time PCR was performed.U6 was used as an internal control of microRNA related samples, and GAPDH was used as an internal control to normalize the differences in the amount of total RNA in other samples.Primers were showed in supplementary Materials.The 2−∆∆Ct method was applied as a statistical tool to calculate relative expression of each gene.The primer sequences are shown in table1.
temperature.Then beads were washed several times in wash buffer and added goat anti-rabbit Immunoglobulin G secondary antibody in antibody buffer for 1 hour at room temperature.The washing steps were repeated and added ChiTag pAG-Tn5 transposon in ChiTag buffer for 1 hour at room temperature.We activated the Tn5 transposase by adding tagmentation buffer containing MgCl2 and incubated for 1 hour at 37°C.The tagmentation reaction were stopped by adding sodium dodecyl sulfate and heating for 10 minutes at 55°C.The tagged DNA fragments were then purified using Tagment DNA Extraction Beads and amplified by PCR using Illumina-compatible primers.Using NovoNGS DNA Cleaning Beads and Qubit dsDNA HS Assay Kit, the PCR products were amplified in reactions containing 33 μl extracted DNA, 2.5 μl primer i5 (N504) and 2.5 μl primer i7 (N704), and 10 μl ×5 AmpliMix buffer.Library sequencing and data analysis were performed by OE Biotech (China).